The sarcoplasmic reticulum (SR) may buffer (attenuate) increases in averaged mycoplasmic free Ca (Ca/m) in quiescent (resting) cells by slowly releasing Ca from the superficial SR to be extruded (unloaded) into the extracellular space. This process is termed "SR Ca unloading" because it results in attenuated agonist-induced ca release. SR Ca unloading suggests close functional association (localization) of SR Ca release sites and sarcolemmal Ca efflux mechanisms, such as the Ca pump and/or Na-Ca exchanger, which would result in subsarcolemmal Ca (Ca/s) localization. We have shown that coronary smooth muscle cells of exercise trained (EX), not sedentary (SED), pigs show SR Ca unloading that is dependent on ryanodine-sensitive Ca release channels. The general hypothesis of this proposal is that the SR in EX cells releases Ca from superficial SR, thus resulting in net Ca efflux (Ca unloading) from the cell with almost no increase in Ca/m. Specific aims are to test the following hypotheses: 1) EX-induced SR Ca unloading requires ryanodine-sensitive Ca release channels and occurs with minimal change in Ca/m, but an increase in free Ca in a subsarcolemmal compartment (Ca/s). Cells will be dispersed from epicardial coronary arteries (conduit size) of EX and SED Yucatan miniature pigs. Whole-cell voltage clamp will allow the use of Ca-activated K current or membrane-bound fura-2 (FFP18) for monitoring Ca/s; mycoplasmic fura-2 salt will monitor Ca/m. 2) EX-induced SR Ca unloading results in net Ca efflux from the cell, not translocation (redistribution) to a caffeine-insensitive store. Net Ca efflux from suspensions of cells will be monitored by extracellular, membrane-impermeant fura-2 3) EX-induced SR Ca unloading requires localized efflux of Ca from the cell at superficial SR sites. The relative localization of Ca efflux from an intact cell will be monitored with digital imaging using extracellular fluo-3 and with an excised (inside-out) patch of Ca-activated K channels used as a "Ca electrode patch". SR locations will be visualized with the green fluorescent SR marker DiOC6. 4) EX-induced SR Ca unloading requires localized Na-Ca exchange. It will be determined whether in EX cells Na- Ca exchange immunofluorescence co-localizes with Ca efflux sites determined with the Ca electrode patch and extracellular fluo-3. These data will provide evidence for a tight functional coupling between slow Ca release from localized, superficial SR sites and Ca extrusion via Na- Ca exchange. 5) SR Ca release is proportional to the contractile response of arterial rings. Simultaneous fura-2 measures of Ca/m and contraction recordings in arterial rings will determine the relevance of SR Ca release to vascular contractile function in intact arteries. The main significance of this work is the integration of functional data obtained from studies at the tissue and subcellular levels of coronary artery smooth muscle.